Septation and separation within the outflow tract of the developing heart

J. Anat.

(2003)

202

, pp327–342

© Anatomical Society of Great Britain and Ireland 2003

Blackwell Publishing Ltd.

REVIEW

Septation and separation within the outflow tract of the developing heart

Sandra Webb,

1

Sonia R. Qayyum,

1

Robert H. Anderson,

3

Wouter H. Lamers

2

and Michael K. Richardson

1

*

1

Department of Anatomy and Developmental Biology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK

2

Department of Anatomy & Embryology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

3

Cardiac Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK

Abstract

The developmental anatomy of the ventricular outlets and intrapericardial arterial trunks is a source of consider-

able confusion. First, major problems exist because of the multiple names and definitions used to describe this

region of the heart as it develops. Second, there is no agreement on the boundaries of the described components,

nor on the number of ridges or cushions to be found dividing the outflow tract, and the pattern of their fusion.

Evidence is also lacking concerning the role of the fused cushions relative to that of the so-called aortopulmonary

septum in separating the intrapericardial components of the great arterial trunks. In this review, we discuss the

existing problems, as we see them, in the context of developmental and postnatal morphology. We concentrate,

in particular, on the changes in the nature of the wall of the outflow tract, which is initially myocardial throughout

its length. Key features that, thus far, do not seem to have received appropriate attention are the origin, and mode

of separation, of the intrapericardial portions of the arterial trunks, and the formation of the walls of the aortic

and pulmonary valvar sinuses. Also as yet undetermined is the formation of the free-standing muscular subpulmo-

nary infundibulum, the mechanism of its separation from the aortic valvar sinuses, and its differentiation, if any,

from the muscular ventricular outlet septum.

Key words

embryo; heart; human; outflow tract; septation.

Introduction

Anomalies involving the outflow channels and their

valves make up a significant proportion of congenital

cardiac defects, with a prevalence of at least 4 per

10 000 births (Ferencz & Neill, 1986; Edmonds & James,

1993). Recent advances in diagnosis and treatment

mean that the majority of these lesions are now ame-

nable to successful surgical correction. It goes without

saying that a sound knowledge of normal development

is essential for the understanding of their morphogenesis.

The mechanisms involved in formation and septation

of the normal outflow tracts and arterial trunks, how-

ever, continue to be controversial. There are several

reasons for the lack of consensus. Some disagreements

merely reflect differences in the techniques used in the

various studies. Others stem from the intrinsic problems

inherent in interpreting the four-dimensional events

that occur during development. Still others reflect

the unequivocal morphological differences that exist

between humans and some of the species used in

experimental studies. Underscoring all these problems

is the plethora of terms used for description of the

processes of development, often with the same term

being used in different fashion by different investigators.

There is also lack of consensus concerning the most

appropriate terms for description of the definitive

ventricular outflow tracts, particularly with regard to

the definition and location of the arterial valvar ‘annulus’.

Correspondence

Sandra Webb PhD, Department of Anatomy and Developmental Biology, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. Fax: +44 20 87250749; e-mail: [email protected]*Current address: Institute of Evolutionary and Ecological Sciences, Leiden University, PO Box 9516, 2300RA Leiden, the Netherlands

Accepted for publication

29 January 2003

Outflow tract of the developing heart, S. Webb et al.


328

So as to set the scene for our developmental discussions

we therefore commence our review with an account

of our understanding of the structure of the intraperi-

cardial components of the ventricular outflow tracts in

the postnatal heart.

The formed heart

The outflow tracts in the definitive heart extend from

the outflow regions of the left and right ventricles

to the margins of the pericardial cavity, where they con-

tinue into the mediastinum as the ascending aorta and

the right and left pulmonary arteries, respectively

(Fig. 1). A key feature within each of these outflow

tracts is the attachment of the leaflets of the arterial

valves. These are hinged in semilunar rather than cir-

cular fashion (Fig. 2), with the semilunar attachments

extending through a significant length of the two

tracts (Anderson, 1990). Distally, the valvar leaflets are

attached at the sinutubular junction (Merrick et al.

2000). This is a true circular boundary that marks the

junctions between the arterial trunks and the more

bulging parts of the arterial roots that form the walls

of the valvar sinuses. Distal to the sinutubular junction,

the intrapericardial components of the arterial trunks

extend to the margins of the pericardial cavity. The

ascending aorta is a solitary trunk within the pericar-

dial cavity, whereas the bifurcation of the pulmonary

trunk into the right and left pulmonary arteries is

within the cavity. The bulging walls of the valvar

sinuses, which are found proximal to the sinutubular

junctions and have an arterial phenotype, also, in the

case of the aortic root, give rise to the left and right

coronary arteries. Proximally, the sinus walls join the ven-

tricular myocardium at the anatomic ventriculo-arterial

junctions. These junctions differ significantly in their

morphology in the right and left ventricles. In the right

ventricle, the anatomic junction between the infundib-

ular musculature and the walls of the valvar sinuses is a

complete circular locus (Fig. 2A). The base of each cup-

shaped valvar leaflet, however, overlaps the anatomic

junction, incorporating a crescent of ventricular myo-

cardium (infundibulum) within the base of each arte-

rial sinus (Fig. 2A,B). The distal attachments of each

leaflet are at the sinutubular junction. Consequently,

three triangles of arterial wall are incorporated within

the right ventricular outflow tract, albeit that each tri-

angle separates the inside of the right ventricle from

extracardiac space. It is the semilunar attachment of

the valvar leaflets that forms the haemodynamic junc-

tion, which straddles the anatomic ventriculo-arterial

junction. When seen in the intact outflow tract there-

fore the circular sinutubular and anatomic ventricu-

loarterial junctions are distinct from the crown-like

haemodynamic junction (Fig. 2C).

Thus, although it possesses the same basic structure

of circular sinutubular and anatomic junctions, with a

crown-like haemodynamic junction, there are signifi-

cant differences in the structure of the aortic as com-

pared with the pulmonary arterial root. Because the

leaflets of the pulmonary valve are supported by a com-

plete sleeve of free-standing infundibular musculature,

they are lifted away from the ventricular base (Merrick

et al. 2000). In contrast, two of the leaflets of the aortic

valve are in continuity posteriorly with one of the leaf-

lets of the mitral (left atrioventricular) valve (Fig. 2D).

This area of fibrous continuity then forms the roof of

the left ventricle. Appreciation of the subtleties of the

structure of the normal outflow tracts is crucial since, to

the best of our knowledge, no account has yet been

Fig. 1 Heart removed from a human cadaver to show the extent of the aorta and the pulmonary trunk within the pericardial cavity. The dashed lines show the distal attachments of the fibrous pericardium.



329

given of the processes involved in formation of the val-

var leaflets, their sinusal attachments and the forma-

tion of the interleaflet triangles, nor the mechanisms of

separation of the intrapericardial parts of the arterial

trunks and the arterial roots. We do not claim to have

solved all these problems ourselves, but their resolution

will be the key to unlocking the remaining mysteries of

the development of the outflow tract.

Nomenclature for the developing outflow tracts

A confusing plethora of terms has been applied to the

different regions of the initially common outflow tract

(Table 1, see also Arráez-Aybar et al. 2003). Numerous

terms have also been employed to account for the

endocardial ridges, or cushions, which divide it. Burg-

gren (1988) highlighted the inconsistent use of the

terms ‘bulbus’ and ‘conus’ to describe parts of the out-

let from the heart in gill-bearing vertebrates. This prob-

lem has also existed over the years with regard to the

description of mammalian cardiac development, and

is further extended by inconsistent use of not only

‘conus’, but also ‘truncus’. Still further problems relate

to the definition of the boundaries between the differ-

ent areas of the developing outflow tract, particularly

as these alter their position during the developmental

period. All of these difficulties are then compounded

Fig. 2 (A) Pulmonary valve from an infant human heart. The valvar leaflets themselves have been removed to show the semilunar mode of their attachments (asterisks). Note the level of the anatomic ventriculoarterial junction between the arterial walls of the pulmonary trunk and the muscular right ventricular infundibulum. (B) Schematic drawing showing the mode of attachment of the valvar leaflets as shown in A, and their relationship to the sinutubular and ventriculoarterial junctions. Note that the bases of the valve leaflets overlap the ventricular myocardium. (C) Relationships of the valvar junctions and the valvar leaflets in three dimensions. Importantly, it demonstrates that the arterial valves have length (double headed arrows). (D) Opened aortic valve from an adult human heart subsequent to removal of the leaflets. As with the pulmonary valve, the leaflets are attached in semilunar fashion, but there is fibrous continuity between the non-coronary and left coronary leaflets of the aortic valve and the aortic leaflet of the mitral valve (double headed arrow). LCA, left coronary artery; RCA, right coronary artery.



330

by the same terms being used in different fashion

between investigators. This is exemplified by the way

different investigators have divided the outflow tract

into ‘truncus’ and ‘conus’, and the way in which they

have described the location of the developing valves

within these components (see Laane, 1978; Pexieder,

1995). Even the popular convention of designating the

area distal to the forming arterial valves as the ‘trun-

cus’, and the area proximal to them as the ‘conus’, is

fraught with difficulty, since the developing valves

themselves, as they form, occupy a significant length of

the developing embryonic outflow tracts. Thus, if the

valvar leaflets are considered to represent the border,

is the segment of outflow tract occupied by the semi-

lunar attachments, between the sinutubular junction

and the base of the leaflets (Fig. 2B), to be considered

as derived from ‘truncus’ or ‘conus’?

In our opinion, if we are to clarify the development

of this important region of the heart, there is a need for

a nomenclature which is explicit and unambiguous, and

which is compatible both with the observed anatomy

of the formed heart and with the dynamic changes seen

during cardiac development. Because of the problems

discussed above, we believe this mandates the use of

descriptive rather than nominative terminology (Fig. 3).

Thus we consider the developing outflow tract as

commencing at the distal extent of the ventricular

loop. The ventricular part of the primary heart tube

itself has a desending inlet component and an ascend-

ing outlet component, from which will grow the apical

components of the definitive right and left ventricles,

respectively (Houweling et al. 2002). It is the compo-

nent of the primary heart tube between the ventricular

loop and the aortic sac that we consider to represent

the outflow tract.

Initially, the common outflow tract is supported

exclusively by the developing right ventricular compo-

nent of the ventricular loop, and is continuous with it.

It extends distally to the margins of the pericardial cav-

ity, where it becomes continuous with the aortic sac

.

Eventually, it will be divided to form the outlets of both

definitive ventricles, and their respective valves, along

with the intrapericardial portions of the arterial trunks.

When first seen, the entire wall of the undivided out-

flow tract, extending to the margins of the newly

formed pericardial cavity, is composed of myocardium.

At these early stages, the presence of a characteristic

acute bend, originally termed the ‘bayonet bend’ by

Orts Llorca et al. (1982), divides the outflow tract into

Tab

le 1

Co

mp

aris

on

of

the

term

ino

log

y u

sed

by

dif

fere

nt

auth

ors

fo

r th

e va

rio

us

reg

ion

s o

f th

e o

utf

low

tra

ct

Kra

mer

(19

42)

Van

Mie

rop

et

al. (

1963

);

Go

or

et a

l. (1

972)

Tan

dle

r (1

912)

; Pe

xid

er (

1978

)A

nd

erso

n e

t al

. (19

74a)

Shan

er (

1962

)La

ane

(197

8)

(ear

ly s

tag

e)La

ane

(197

9)

(lat

e st

age)

Qay

yum

et

al. (

2001

)

aort

ic s

acao

rtic

sac

aort

ic s

acao

rtic

sac

ven

tral

ao

rta

aort

ic s

actr

un

cus

mes

ench

ymal

isao

rtic

sac

tru

ncu

s ar

teri

osu

str

un

cus

dis

tal b

ulb

us

tru

ncu

s

bu

lbu

s

dis

tal s

egm

ent

tru

ncu

s ar

teri

osu

s (m

yoca

rdia

l se

gm

ent)

dis

tal s

egm

ent

con

us

cord

isco

nu

sp

roxi

mal

bu

lbu

sd

ista

l bu

lbu

sm

idd

le s

egm

ent

pro

xim

al s

egm

ent

Pro

xim

al

seg

men

t

5 4 6 4 7

1 4 2 4 3

5 4 6 4 7

1 4 2 4 3



331

proximal and distal parts (Fig. 4). We now describe this

characteristic landmark as the ‘dog-leg’ bend. This def-

inition of proximal and distal parts of the outflow tract

follows the precedent established by Ya et al. (1998),

and has recently been adopted by Yelbuz et al. (2002).

Early development of the primary heart tube

The heart is one of the first organs to form in amniotes.

The bilateral and symmetrical cardiac primordia

migrate to the midline, where they form the primary

heart tube. This initially tubular heart consists of an

inner endothelial layer, the endocardium, and an outer

muscular layer, the myocardium. Contrary to conven-

tional wisdom, not all regions of the developing heart

are present at the initial stages of development. Line-

age studies from our laboratory (N. Brown, personal

communication) have shown that, in the mouse, the

initial straight part of the tube becomes the left ven-

tricular component of the definitive heart, with the

other segments being recruited at a later stage. De La

Cruz et al. (1991), summarizing their earlier work (De

La Cruz et al. 1977), showed that, in the chick, the com-

parable straight part of the tube formed the apical

trabecular component of the right ventricle. More

recent studies have shown that the entirety of the

developing outflow tract, along with the developing

right ventricle, receives a contribution from a second-

ary, or anterior, heart field (Kelly et al. 2001; Mjaatvedt

et al. 2001; Kirby, 2002). This origin from a secondary

source accounts for the lengthening of the outflow

Fig. 3 This diagram of the developing embryonic heart illustrates our suggested terminology. Note that we define proximal and distal segments of the outflow tract. In the definitive situation, as shown in Fig. 4, the boundary between these components is marked by the characteristic bend ‘dog-leg’ bend. The bend has been ignored for the purposes of this drawing. The boundary between the distal outflow segment and the arterial segment, the aortic sac, is at the level marked by the reflections of the pericardial cavity (see also Fig. 1).

Fig. 4 This scanning electron micrograph of a human embryo, at Carnegie stage 15 (approximately 34 days of gestation), shows a ventral view of the heart. The distal and proximal segments of the unseptated outflow tract are separated by a characteristic dog-leg bend. The proximal segment lies across the atrioventricular junction.



332

tract myocardium up to Hamburger Hamilton stage 21

in the chick (Rychter, 1978; Mjaatvedt et al. 2001), to

embryonic day 9.5 in the mouse (Kelly et al. 2001), and

to embryonic day 11 in the rat (Ya et al. 1998). This cor-

responds to Carnegie stage 13 in the human. Impor-

tantly, this contribution to the outflow tract from the

secondary heart field is complete by the time the out-

flow cushions are invaded by cells derived from the

neural crest (Le Douarin, 1982; Kirby et al. 1983; Fukii-

shi & Morriss-Kay, 1992; Jiang et al. 2000).

The structure of the distal outflow tract

In the human, at Carnegie stage 14, the myocardial wall

of the outflow tract extends to the border of the peri-

cardial cavity (Fig. 5A), where it joins the aortic sac, the

latter giving rise to the arteries that feed the pharyn-

geal arches. Eventually, this common outflow tract will

give rise not only to the valves and sinuses of both definit-

ive ventricular outflow tracts, but also to the intraperi-

cardial portions of the arterial trunks (shown at Carne-

gie stage 16 in Fig. 5B). Our observations in the human,

to be illustrated below, confirm those made by Ya et al.

(1998) in the rat, showing that the definitive sinutubu-

lar junctions are formed at the level of the developing

outflow tract marked initially by the dog-leg bend.

Most previous studies have suggested that it is elon-

gation and division of the aortic sac that produces the

intrapericardial parts of the arterial trunks, these being

distal to the dog-leg bend. The cushions initially found

within the distal part of the muscular outflow tract are

said to separate the developing aortic and pulmonary

roots, with the proximal cushions separating the ven-

tricular outflow tracts (Van Mierop, 1979). If correct,

this process would necessitate the proximal displace-

ment of the cushions from a position initially distal to

the dog-leg bend. Our own observations do not sup-

port these interpretations. Instead, we believe that it is

the distal cushions that divide the distal outflow tract

into the intrapericardial parts of the aorta and pulmo-

nary trunk, with the proximal cushions separating both

the arterial roots and their ventricular outflow tracts.

In the light of these differences in interpretation, it is

pertinent at this stage to provide a brief review of the

various previous concepts.

Previous concepts of septation of the outflow tract

Van Mierop et al. (1963) and Van Mierop (1979) sug-

gested that three components were needed to divide

the outflow tract, namely the aortopulmonary septum,

along with separate sets of distal and proximal ridges.

According to this concept (Fig. 6), the ridges initially

fuse distally to form a septum, which then fuses with

Fig. 5 (A) Section through the outflow tract of a human embryo at Carnegie stage 14 (approximately 36 days of gestation) sectioned in the sagittal plane. It shows how the distal outflow tract with its myocardial wall (m) extends to the edge of the pericardial cavity, where it becomes continuous with the aortic sac. The aorto-pulmonary septum is seen as a wedge of tissue in the posterior wall of the sac (asterisk), separating the origins of the fourth and sixth aortic arches (4,6), the latter seen giving rise to one pulmonary artery. The outflow tract is being septated by the distal cushions. (B) Section from a human embryo at Carnegie stage 16 (approximately 37 days of gestation), which has been sectioned in the transverse plane. The distal cushions (asterisk) are shown separating into the intrapericardial parts of the aorta and the pulmonary trunk. Note the incipient arterialization of the walls of these trunks. The proximal outflow tract has retained its myocardial phenotype, proximal to the arrowheads.



333

the aortopulmonary septum, the latter growing down

from between the fourth and sixth pharyngeal arches.

Septation then proceeds with fusion of the proximal

ridges to form a proximal conal septum, which then

combines with the proximal end of the distal septum to

complete the process of septation.

Icardo (1990), in contrast, promoted the concept of

formation of a spiral septum. Like Van Mierop et al.

(1963), he claimed to recognize separate proximal and

distal sets of ridges, but he argued that spiralling was

achieved by end-to-end fusion of each of the paired

proximal ridges with an opposite partner of the paired

distal ridges (Fig. 7A). This, he suggested, produced

conjoined ridges, which then fused lengthwise, cross-

ing at their midpoints.

Several other groups of investigators have illustrated

formation of a spiral septum but, in their view, the sep-

tum is formed by fusion of two continuous longitudinal

ridges (Fig. 7B). These ridges are described as taking a

spiralling course as they extend through the length of

the outflow tract (Kramer, 1942; Anderson et al. 1974a;

De La Cruz et al. 1977; Pexieder, 1978). Some of those

who espoused this model also emphasized that it

required subsequent ‘detorsion’ of the separated arte-

rial pathways (Fig. 7C,D).

Bartelings & Gittenberger-de Groot (1989), having

studied human hearts, then offered yet another

hypothesis. This placed even greater emphasis on

growth of a structure they call the aortopulmonary

septum (Fig. 8). According to their concept, septation

of the outflow tract is achieved with little or no contri-

bution from the endocardial ridges.

Over time therefore some have described separate

ridges in the proximal and distal segments, while

others have accounted for two elongated ridges that

extend throughout the entire length of the outflow

tract. But even those who have described separate sets

of proximal and distal ridges have then disagreed on

Fig. 6 Model of development whereby three components, namely the distal cushions, the proximal cushions and the aortopulmonary septum (AP septum), contribute to the septation of the outflow tract. Only those components that give rise to the septal structures are shown, the valvar leaflets having been excluded for simplicity. The panels show the presumed steps in septation.

Fig. 7 Models for spiral septation of the outflow tract. The arrows in panel A indicate the fusion of opposite distal and proximal cushions to form spiralling longitudinal ridges, as shown in panel B. Others, however, argue that the spiralling ridges exist through the length of the outflow tract from the outset (see text for discussion). Be that as it may, having fused to form a spiralling septum, it is argued that ‘detorsion’ (panel C) is required to produce the definitive septum (panel D).

Fig. 8 Diagram illustrating the concept of separation of the outflow tracts primarily by the aortopulmonary septum. Panel A shows the distal cushions fusing with the aortopulmonary septum to form a common septal structure (panel B). By the stage shown in C, the proximal cushions remain only as the posterior wall of the subpulmonary infundibulum (grey cube), with the aortopulmonary septum having reduced to a remnant of tissue external to the heart (cross-hatched).



334

the number of ridges to be found distally, particularly

with regard to the situation in the chick. Tonge (1869)

described only two ridges proximally in the developing

chick heart, but illustrated three ridges distally. Laane

(1978), and Waldo et al. (1998), confirmed the exist-

ence of the three distal ridges, but Laane argued that

two of the ridges fused cranially to form a common

ridge, with the ridges remaining unfused caudally. Los

(1978), in contrast, showed only two longitudinal

ridges in his illustrations of the developing chick heart,

extending throughout the length of the outflow tract.

Using scanning electron microscopy, supported by

reconstructions from serial sections, Qayyum et al.

(2001) confirmed the initial opinion of Tonge (1869),

demonstrating two ridges proximally, but three ridges

distally. This avian arrangement parallels the situation

in the developing reptilian heart, for which Shaner

(1962) described a tripartite distal arrangement. To the

best of our knowledge, this third distal ridge has never

been seen in the developing mammalian heart. The

three ridges seen distally in the chick are found in addi-

tion to the so-called intercalated cushions. The third

distal ridge develops after the first two, but before the

intercalated cushions. The intercalated cushions are

seen level with the developing valves, and do not

extend below them. Reinforcing the opinion first

expressed by Tonge (1869), it is our belief that the

intercalated cushions form one leaflet, with its support-

ing sinus, for each of the aortic and the pulmonary

valves. Unlike most of the accounts summarized above,

nonetheless, it is also our belief that the function of the

cushions found in the distal outflow tract is to divide

this part of the developing heart into the intrapericar-

dial parts of the aorta and pulmonary trunk, the cush-

ions themselves subsequently disappearing as the

arterial trunks separate one from the other. At this

stage therefore we will review briefly our own recent

findings, supplementing them with illustrations of

developing human hearts.

Formation of the intrapericardial arterial trunks

When first formed, the junction between the distal end

of the outflow tract and the aortic sac is found at the

distal extent of the pericardial cavity. Within the heart,

this corresponds with the distal extent of the endocar-

dial ridges or cushions (Fig. 5A). As already discussed,

the walls of this distal part of the outflow tract,

between the dog-leg bend and the aortic sac, possess a

myocardial phenotype. It has still to be determined

how this distal outflow tract, within the pericardial cav-

ity, becomes converted to the intrapericardial compo-

nents of the aortic and pulmonary trunks. Ya et al.

(1998), having studied the rat heart, argued that this

transformation was the consequence of transdifferen-

tiation of the walls from a myocardial to an arterial

phenotype. Arguello et al. (1978) had earlier proposed

this concept, following their ultrastructural investiga-

tions of the developing outflow tract of the chick. It is

possible, however, that cells from the pharyngeal mes-

enchyme migrate into the walls of the distal outflow

tract, replacing the myocardial cells. The mechanism

underscoring this crucial change therefore has still to

be determined.

Clarification of the mechanism by which the intra-

pericardial parts of the arterial trunks are separated from

one another, nonetheless, can help decide whether

they are derived from the distal outflow tract or the

aortic sac. According to our observations, septation

of this area is achieved by fusion of the distal cushions

(Fig. 5B). During this process, the cushions themselves

seemingly disappear, with the aorta and pulmonary

trunk developing their own walls (Fig. 9A,C). The tissue

between the newly forming arterial trunks, which

was initially continuous with the posterior pharyngeal

mesenchyme (Fig. 5B), will eventually disappear as space

is produced between the intrapericardial parts of the

aorta and pulmonary trunk (Fig. 1).

The traditional concept for separation has been that

the intrapericardial arterial trunks are separated by

downgrowth of the so-called aortopulmonary septum,

albeit that there have been various definitions for this

septal complex. Our observations in the human heart

show that the initial ‘aortopulmonary septum’ is no

more than a relatively insignificant wedge of tissue

interposed between the origins from the aortic sac of,

on the one hand, the arteries supplying the third and

fourth arches and, on the other hand, the arteries

which feed the developing sixth arches (Fig. 5A). These

observations endorse the concept of structure of the

aortopulmonary septum as shown in the reconstruc-

tion of the aortic sac made by Steding & Seidl (1990).

This structure at the site of bifurcation appears to be

more extensive in the chick. We suggest that this

reflects the topographical differences known to exist in

this area of the chick as compared to mammals. As far

as we are aware, none of those arguing for separation



335

of the arterial trunks by this ‘aortopulmonary septum’

have considered how the purported ‘septal’ structure

subsequently loses its septal role concomitant with the

development of separate walls for the intrapericardial

arterial trunks. This loss of an embryonic septal role is

crucial for understanding not only the division of the

distal outflow tract, but also its proximal components.

Separation of the proximal outflow tracts

Our most recent studies, conducted in mammals and

birds, suggest that the basic mechanics of separation

are comparable in both parts of the outflow tract,

when cognisance is taken of the difference in morphol-

ogy of the distal component. As explained above, most

agree that the longitudinal cushions within the out-

flow tract can arbitrarily be divided into proximal and

distal components at the dogleg bend. Initially, the

opposing cushions fuse across the lumen of the out-

flow, with fusion starting distally and proceeding prox-

imally. Also, as discussed above, it is our belief that the

major function of the distal cushions is to separate the

distal common outflow tract into the aortic and pulmo-

nary components of the intrapericardial arterial trunks.

And, as explained, it has still to be established how the

walls of the intrapericardial trunks achieve their arte-

rial phenotype, a process that occurs with remarkable

rapidity. The distal cushions, nonetheless, having per-

formed their septal function at the early stages of

development, subsequently disappear by a mechanism

again as yet unknown. Irrespective of the mechanisms,

the aortic and pulmonary trunks are subsequently seen

as separate structures, each with its own discrete walls,

within the pericardial cavity (Figs 1 and 9C).

subsequent to the septation of the distal outflow tract. Now the distal parts of the proximal cushions, together with the intercalated cushions, are cavitating (arrowheads) to form the valvar leaflets of the aorta (Ao) and the pulmonary trunk (PT), along with the walls of their supporting sinuses. Note the different stages of arterialization of the different sinusal walls, and note also that the sinuses remain enclosed within the myocardial cuff, through which the left (LCA) and right (RCA) coronary arteries are penetrating to enter the valvar sinuses. The dark staining fibrous tissue (asterisk) marks the initial site of fusion of the distal parts of the proximal cushions. (C)

A more cranial slide from the same embryo, which shows that, by this stage, the intrapericardial parts of the arterial trunks are separate structures, with a bar of fibro-adipose tissue now occupying the former site of the distal cushions.

Fig. 9

(A)

Frontal section through a human embryo at Carnegie stage 14 (approximately 35 days of gestation). The dorsal outflow tract has been separated into the interpericardial parts of the aorta (Ao) and the pulmonary trunk (PT). The proximal cushions (asterisks) have yet to fuse, but the dense mesenchymal tissue that originates from the neural crest is penetrating both cushions. RAp, LAp: right and left atrial appendages; LV, left ventricle. (B)

Transverse section through a human embryo at Carnegie stage 22 (approximately 54 days of gestation) shows the stage



336

The sequence of separation seen distally (Fig. 9A) is

then replicated within the proximal outflow tract. This

proximal part, separated from the distal outflow tract

by the dog-leg bend, itself has distal and proximal com-

ponents. The distal part of this proximal outflow tract,

immediately upstream of the dog-leg bend, is divided

by fusion of the distal ends of the proximal cushions.

These cushions, along with the intercalated cushions

that have by now appeared within the outflow tract

(Fig. 10), form the leaflets and supporting sinusal walls

of the aortic and pulmonary valves. One of the interca-

lated cushions forms a leaflet and sinus of the aortic

valve, while the other intercalated cushion forms the

comparable components of the pulmonary valve. The

other two leaflets and sinuses of each arterial valve are

derived from the cushions that fused to septate this

distal part of the proximal outflow tract. Each fused

cushion, on its opposite face, gives rise to two valvar

leaflets, one for the aortic and the other for the pulmo-

nary valve (Fig. 9B). By a process as yet unknown, the

cushions then undergo a remodelling, or cavitation, to

form the definitive cup-shaped valvar leaflets along

with their sinusal walls. The appearance of the cavities

separates the cushions themselves into luminal and

mural components. The luminal parts become the

valvar leaflets, while the mural parts arterialize to form

the walls of the supporting valvar sinuses. As part of

this process, the distal part of the proximal cushions,

like the distal cushions, must also lose their septal func-

tion, thereby separating the arterial roots.

At the start of this process of fusion, the developing

arterial roots are completely encased within a myocar-

dial cuff (Fig. 9B). Gradually, as demonstrated by Ya

et al. (1998) in the rat, and endorsed by Rothenberg

et al. (2002) in the chick, this cuff disappears. Concom-

itant with its disappearance, a plane of fibro-adipose

tissue is formed at the centre of the cushions, eventu-

ally becoming continuous with the extracardiac space,

and then separating the aortic from the pulmonary

root (Fig. 9C).

Fig. 10 Reconstruction made from a human heart of 5 weeks gestation (Carnegie stage 15), viewed from the ventral aspect, soon after immigration of neural crest cells has begun. The left-hand panel shows the overall arrangement, with the arrangement of the individual cushions shown to the right. (A,C) Intercalated ridges or cushions, which occupy the area of the dog-leg bend; (B) septal outflow ridge; (D) parietal outflow ridge. Localized accumulations of neural crest-derived mesenchyme form ‘prongs’, shown in purple. Note that they are located within the distal parts of the cushions of the proximal outflow tract, being positioned just proximal to the dog-leg bend.



337

The most proximal parts of the cushions within the

proximal component of the outflow tract then also

fuse to form a structure that, at first, is a septum within

the ventricular outflow tract (Fig. 11A). When this

structure is first formed, the developing right ventricle

supports the entirety of the outflow tract. This newly

formed embryonic outlet septum therefore is exclu-

sively a right ventricular structure. Its free edge over-

rides the cavity of the right ventricle. The endocardial

cushion tissue forming this proximal embryonic outlet

septum subsequently becomes muscular. This process,

known as ‘myocardialization’, is the consequence of

invasion of the cushions by cardiac myocytes pre-

existing within the parietal walls of the outflow tract

(Okamoto, 1980; McBride et al. 1981; Okamoto et al.

1981; Lamers et al. 1995; Van Den Hoff et al. 1999). As

the partition, now muscularized, fuses with mesen-

chyme crowning the crest of the muscular ventricular

septum, it walls the aorta into the left ventricle. At the

same time, the muscular partition itself becomes the

supraventricular crest of the right ventricle, which then

separates the cavity of the right ventricle from the

aortic valvar sinuses (Fig. 11B). In postnatal life there-

fore none of the structures that initially divided the

embryonic outflow tract into aortic and pulmonary

components continues to occupy a septal position.

Fig. 11 (A) Sagittal section from a human embryo at Carnegie stage 20 (approximately 50 days of gestation). The proximal cushions of the outflow tract have fused to form an embryonic outlet septum within the right ventricle (asterisk). The interventricular foramen, which links the right ventricle to the subaortic outflow, is seen as a channel positioned caudal to this outlet septum. The distal outflow segment has separated into the aortic and pulmonary trunks, each having an arterial phenotype. (B) Sagittal section from a human embryo of 11 weeks gestation. An extracardiac bar of tissue is now seen between the walls of the pulmonary trunk and the sinuses of the aorta (arrowheads). The proximal cushions have now myocardialized to form the subpulmonary infundibulum (asterisk). Abbreviations: Ao, aorta; LA, left atrium; PT, pulmonary trunk; RV, right ventricle.



338

Rotation of the outflow tracts

In the definitive heart, the pulmonary trunk unequivo-

cally spirals round the aorta, from the off-setting of the

arterial valves proximally to the separate arterial trunks

distally (Merrick et al. 2000). In terms of development,

several groups of investigators (Kramer, 1942; Patten,

1953; Van Mierop et al. 1963; de La Cruz et al. 1977;

Pexieder, 1978) have previously argued that the rota-

tion of the outflow tract, and the concomitant spiral-

ling of the ridges, are the consequence of events

occurring as part of the process of looping. Other

groups (Anderson et al. 1974a; Goor et al. 1972; Los,

1978; Laane, 1979) agreed concerning rotation, and

also agreed that the event would generate primary tor-

sion. These investigators, however, argued that such

primary torsion would need to be followed subse-

quently by anticlockwise rotation distally, thus causing

‘unwinding’ or ‘detorsion’ of the spiral pattern (Fig. 7).

They stated that the ‘detorsion’ was then transferred to

the arterial trunks, thus establishing the adult spiral

configuration. Still others, notably De La Cruz & Da

Rocha (1956) and Steding & Seidl (1980, 1981), argued

that they were unable to find evidence of rotation of

the ventricular outlets during normal development.

Our current findings endorse the lack of active rota-

tion subsequent to the initial formation of the outflow

tract. Indeed, we are now able to explain why there is

no need to propose ‘detorsion’ as part of the mecha-

nisms of septation. When first formed, the endocardial

ridges themselves have an unequivocally spiral path

within the outflow tract (Fig. 10). As the ridges fuse dis-

tally, there is concomitant arterialization of the sepa-

rated aortic and pulmonary pathways (Fig. 9A). Thus,

the structure that initially was a common distal outflow

tract, divided by a spiralling septum, is replaced by sep-

arate arterial trunks that spiral round each other as

they leave the cardiac base (Fig. 1). The retraction of

the myocardial wall of the outflow tract, as it assumes

a distal arterial phenotype, therefore serves simply to

reveal the now separate, but still spiralling, aortic and

pulmonary trunks.

Reduction of the inner heart curvature

It has often been stated that, during normal develop-

ment, there is a marked shift in the position of the sub-

aortic outlet. Initially, this proximal and posterior

(dorsal) part of the outflow segment, which eventually

becomes incorporated into the left ventricle, is sup-

ported exclusively by the developing embryonic right

ventricle, itself formed from the distal part of the ven-

tricular loop. Goor et al. (1972) argued that absorption

of the proximal segment into the left ventricle was

secondary to a process of migration, which carried the

aorta over the left ventricle. Anderson et al. (1974a,b),

in contrast, suggested that the process of absorption

was primary, and that transfer of the aorta to the

definitive left ventricle occurred concomitant with the

reduction of the tissue that formed the inner heart

Fig. 12 Section from a human embryo at Carnegie stage 17 (approximately 44 days of gestation), sectioned in the sagittal plane. It shows that, although the aortic valve is being sequestered within the left ventricle by fusion of the proximal parts of the outflow cushions to the crest of the muscular ventricular septum, the musculature of the inner heart curvature (dashed line) still separates the developing leaflets of the aortic and mitral valves. The subaortic outlet is marked by an asterisk. The arrowheads indicate the atrioventricular endocardial cushions. AO, aorta; PT, pulmonary trunk; LA, left atrium.



339

curvature. The muscular curve itself has variously been

termed the ‘conoventricular flange’ (Kramer, 1942),

or the ‘bulboatrioventricular flange’ (Anderson et al.

1974a,b).

The flange forms the roof of the most direct route, in

the embryonic heart, from the ventricles to the subaor-

tic outlet, which unequivocally becomes an integral

part of the left ventricle (Fig. 12). The finding of other-

wise normal hearts, but with muscular tissue separating

the leaflets of the aortic and mitral valves (Rosenquist

et al. 1976), shows that complete reduction of the inner

curve is not a prerequisite for complete transfer of

the aorta to the left ventricle. The inner curvature,

nonetheless, does undergo structural change during

development, but this change takes place after the

completion of cardiac septation, and without disturb-

ing the topographic arrangement. Thus, examination

of human embryos shows that at the time of fusion of

the endocardial cushion tissue surrounding the embry-

onic interventricular foramen, a process which walls

the aorta into the left ventricle and incorporates

part of the primary ventricular foramen as the left ven-

tricular outflow tract, a considerable portion of the

myoblastic tissue of the inner curve remains interposed

between the developing leaflets of the aortic and

mitral valves (Fig. 12). Only much later in development

does this muscular tissue become converted into the

area of fibrous continuity seen between these leaflets

as a characteristic feature of the normal left ventricle

(Fig. 2D). The mechanism of disappearance of this

musculature of the inner heart curvature has still to

be established.

Formation of the subpulmonary infundibulum

Bartelings & Gittenberger-de Groot (1989) argued that

invasion of the proximal outflow cushions by the limbs

of the structure they call the aortopulmonary septum

provided the stimulus for mobilization of myocardium

to form the posterior wall of the free standing subpul-

monary infundibulum. We, too, have been able to

trace the ‘prongs’ of neural crest-derived condensed

mesenchyme into the proximal cushions (Fig. 10). Their

position marks the eventual site of septation of the

proximal outlet into the aortic and pulmonary roots.

We have not, however, been able to trace the rods

from the true aortopulmonary septum, namely the

wedge of tissue situated between the origins of the

fourth and sixth arch arteries from the aortic sac.

Indeed, we do not place great emphasis on this struc-

ture contributing to division of the outflow tract. In our

opinion, it is more important to note that, when the

most proximal parts of the cushions have fused to sep-

tate the proximal outflow tract, they subsequently lose

their septal function, concomitant with the process of

muscularization and formation of the free-standing

subpulmonary muscular sleeve. Part of this process is

the formation of space between the posterior wall of

the subpulmonary infundibulum and the anterior

sinuses of the aortic root (Fig. 13).

The space thus formed is continuous with the space

that develops between the intrapericardial parts of

the great arterial trunks (Fig. 9C). Completion of the

Fig. 13 This dissection of an adult human heart shows the relationship of the definitive ventricular outflow tracts. The arterial trunks have been removed, and the base of the heart is viewed from above. The anatomic pulmonary–ventricular junction, between the arterial wall of the pulmonary trunk (PT) and the muscular right ventricular infundibulum, is shown by the line of asterisks. Note the deep tissue plane (white dashed line) that separates the free-standing subpulmonary infundibulum of the right ventricle from the wall of the right coronary sinus of the aorta. Note also the left coronary artery. LCS, RCS, NCS – left, right, and non-coronary sinuses of the aorta, respectively.



340

process of separation depends on disappearance of the

myocardial cuff that initially surrounded the entirety of

the distal part of the proximal outflow tract (Fig. 9B).

These changes explain fully the postnatal morphology

of the outlet from the right ventricle, which is muscular

over its entire circumference, forming a cylindrical

sleeve that can be removed without encroaching the

cavity of the left ventricle (Stamm et al. 1998; Merrick

et al. 2000). Apoptosis is thought to be involved both in

retraction of the myocardial sleeve of the proximal out-

flow tract (McBride et al. 1981) and in triggering the

subsequent myocardialization of the proximal septum

(Poelmann et al. 1998). It has also been suggested that

the latter process may involve signalling by growth fac-

tors (Sanford et al. 1997). The precise mechanism of

formation of the free-standing infundibulum, and its

separation from the aortic root, however, currently

remains unexplained.

Cardiac neural crest

Many investigators, such as Phillips et al. (1987) and

Jiang et al. (2000), have now confirmed the initial stud-

ies of Kirby et al. (1983, 1985), namely that cells migrat-

ing into the heart from the cardiac neural crest are

crucially important in septation of the outflow tracts.

Having entered the outflow tract, the cells form two

conspicuous structures, often interpreted as ‘limbs’ of

the aortopulmonary septum (Laane, 1978; Los, 1978;

Thompson & Fitzharris, 1979; Thompson et al. 1983,

1984; Bartelings, 1990). To the best of our knowledge,

it has never been shown that the limbs are in continuity

with the tissues separating the origins of the arteries

supplying the fourth and sixth pharyngeal arches from

the aortic sac, this tissue, as far as we can see, represent-

ing the initial aortopulmonary septum. We have con-

firmed that the cells from the neural crest enter the

distal ridges directly, but we have found that, when the

ridges fuse, a characteristic whorl of condensed mesen-

chyme is found at the junction between the distal and

proximal parts of the outflow tract (Fig. 9A). Extensions

from this whorl, the rods or prongs, run proximally

within the fusing ridges (Fig. 10). These rods play a

major role in dividing the distal part of the proximal

outflow tract into separate aortic and pulmonary

valves and their respective ventricular outflow tracts.

This is confirmed by experimental ablation of the

cardiac neural crest, which is known to lead to a variety

of malformations involving the arterial roots, often

producing a common arterial trunk (Kirby, 1983; Kirby &

Bockman, 1984; Kirby et al. 1985; Nishibatake et al.

1987). It is known that the cells from the crest reach

proximally as far as the arterial valvar leaflets (Taka-

mura et al. 1990). The contribution cannot be major,

however, since ablation of the neural crest has little

effect on the formation of the valvar leaflets. It remains

to be established with certainty whether the cells from

the neural crest migrate further proximally into the

heart itself (Takamura et al. 1990; Noden et al. 1995;

Creazzo et al. 1998; Poelmann et al. 1998; Waldo et al.

1998). There is also uncertainty whether the cells

derived from the crest persist as septal elements or are

eliminated by apoptosis (Icardo, 1990; Poelmann et al.

1998; Jiang et al. 2000).

Conclusions

Many of the persisting problems concerning the devel-

opment of the ventricular outflow tracts reflect the dif-

ficulties inherent in making correlations between the

structure of the outflow tracts during their develop-

ment and their definitive morphology. Knowledge of

the fate of this developing area is pivotal to the under-

standing of the formation of the definitive ventriculo-

arterial junctions. Crucially, these junctions shift

markedly during development of the ventricular outlets.

Initially, the anatomic ventriculo-arterial junction is

the border between the distal end of the ventricular

outflow segment and the aortic sac. This is positioned

at the margins of the pericardial cavity. In the definitive

heart, separate ventriculo-arterial junctions are found

within the right and the left ventricles towards the

bases of the arterial valves. The distal attachments of

the leaflets of the arterial valves are at the sinutubular

junctions. These circular landmarks are formed at the

site of the dogleg bend, which initially separates the

two parts of the muscular outflow tract. The valvar

leaflets, along with their supporting arterial sinuses

therefore are formed distally within the proximal out-

flow tract. The most proximal part of the outflow tract

persists largely as the subpulmonary infundibulum, the

aortic vestibule becoming fibrous posteriorly subse-

quent to disappearance of the musculature of the inner

heart curve. The definitive anatomic ventriculo-arterial

junctions are eventually located at markedly different

levels within the right and left ventricles. It is the

formation of the free-standing infundibular sleeve of

the right ventricle, by muscularization of the proximal



341

cushions, that largely accounts for the differences in

these levels in the right as opposed to the left ventri-

cles. The developmental mechanisms producing all

these changes have still to be clarified.

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